Autoimmune diseases are characterized by an abnormal immune response (involving either immune system cells or antibodies) directed against normal autologous (self) tissues. Autoimmune diseases afflict huge numbers of individuals throughout the world.
A normal immune system has the capacity to identify and destroy a large variety of foreign invader organisms such as bacteria and viruses. Remarkably, a normal immune system can readily distinguish foreign substances from self, and thereby is able to react vigorously against potentially pathogenic entities from the environment without harming the host's own cells.
The immune system's non-reactivity to self is termed immunological tolerance. In pathological situations, immunological tolerance to a wide variety of self substances is broken, resulting in an autoimmune response. If of an appropriate nature and of sufficient severity and duration, the anti-self response will result in an autoimmune disease. Cellular immune mechanisms are believed to be primarily involved in insulin dependent diabetes (IDD).
Whereas susceptibility to autoimmune diseases may be inherited through the defective actions of multiple genes, indirect evidence suggests that an interaction with a foreign substance from the environment may also be necessary to induce the pathogenic process that results in disease. One explanation for this is that immunization with the foreign inductive chemical induces a cross-reactive response to self through molecular mimicry or chemical similarity. However, once the autoimmune process has been initiated, other secondary immunizing events involving other self antigens typically occur through the release of intracellular constituents in forms not normally encountered by the immune system. Targeted organs thus become damaged through the combination of all of these events, which leads to the appearance of a clinically recognized disorder only when the disease process has progressed to ablate large numbers of tissue cells so targeted.
A number of strategies have been used or proposed to suppress autoimmune diseases, most notably drugs, such as cyclophosphamide, cyclosporin A, methotrexate, and azathioprine. Steroid compounds, such as prednisone and methylprednisolone, are also employed in many instances. These drugs have limited long term efficacy against both cell- and antibody-mediated autoimmune diseases. Use of drugs is limited by virtue of their toxic side effects which include universal immunosuppression. Prolonged treatment with these drugs inhibits the normal protective immune response to pathogenic microorganisms, thereby increasing the risk of infections. A further drawback is that immune-mediated elimination of aberrant cells is impaired and there is, thus, an increased risk that malignancies will develop in patients receiving prolonged global immunosuppression.
Because the subject invention concerns insulin dependent (Type I) diabetes, a detailed background of diabetes is provided below.
Insulin dependent diabetes
Diabetes mellitus comprises a group of diseases that result in elevation of the blood glucose level because of relative or absolute deficiency in the pancreatic hormone insulin. Insulin is secreted into the blood when food is ingested and primarily directs absorbed nutrients into body stores. Diabetes is a major public health problem affecting at least 5 million and as many as 10 million Americans. The prevalence of the most severe form of IDD is 1 in 300 in the United States.
Chronic elevation of the blood glucose level is the most obvious metabolic effect in diabetes and is associated with progressive damage to blood vessels. This may lead to heart attack, stroke, blindness, peripheral nerve dysfunction, and kidney failure. The frequency and severity of diabetes-related complications are greatest in the insulin dependent form of the disease, in which an immunological destruction of the insulin secreting pancreatic beta cells occurs. The high rate of irreversible complications in IDD occurs despite the availability of insulin replacement through injections given 1-4 times daily.
Insulin and other pancreatic hormones are well known and characterized. See, for example, Steiner et al. (1989) "Chemistry and Biosynthesis of Pancreatic Protein Hormones," in Endocrinology; DeGroot et al., EDs., W.B. Saunders Company, p. 1263-1289. As described in Steiner et al., the amino acid sequence of insulin is highly conserved across a number of species, including human, monkey, swine, and ox. IDD has proved itself to be predictable both in unaffected relatives of patients with IDD, as well as in persons from the general population. A predisposition to develop clinical diabetes can be detected through several different tests. For example, genetic susceptibility to diabetes has become increasingly definable through the use of molecular biological means, usually from DNA samples obtained from peripheral blood. One major gene involved in the inherited susceptibility to IDD is that located at the HLA-DQ locus. It is currently possible to identify risks varying from essentially none to those as high as 70 fold above those without the genotype. In families a genetic risk as high as 1 in 4 can be estimated for unaffected siblings just through identification of HLA haplotypes shared with the affected proband.
Persons who have just developed IDD or are in process of developing IDD have a number of disease-specific autoantibodies in their blood. Such autoantibodies include those to islet cell antigens (ICA), to beta cell specific proteins of 64 kDa, which are now believed to be the lower molecular isoform of glutamic acid decarboxylase (GAD.sub.65), to native insulin and proinsulin, and to a number of more minor determinants such as carboxypeptidase-H and heat shock proteins belonging to the hsp-60 family.
Insulin autoantibodies (IAA) are observed in untreated, newly diagnosed IDD patients (Palmer et al. (1983) Science 222, 1337-1339) as well as in apparently unaffected relatives of diabetic probands. Whereas autoimmunity to insulin could directly cause beta-cell damage, could interfere with the action of endogenous insulin, or could have both effects, some investigators suggested that IAA reflect the rate of islet cell destruction and thus act merely as reporters of aggressive islet directed autoimmunity (Ziegler et al. (1989) Diabetes 38, 1320-1325; Vardi (1988) Diabetes Care 11, 736-739).
The spontaneously diabetic non-obese diabetic (NOD) mouse and the BB rat are useful animal models for human IDD. Analysis of these animals provides important insights into the sequence of pathogenic events, and leads to an understanding of the nature of the autoimmunological process. Previous studies from several laboratories have demonstrated that an extended prophylactic course of daily, subcutaneous injections of high doses of insulin protected NOD mice and BB rat from both hyperglycaemia and islet infiltration by mononuclear leucocytes (insulitis) (Atkinson et al. (1990) Diabetes 39, 933-937, Gotfredsen et al. (1985) Diabetologia 28, 933-935).
In addition, prophylactic treatment with high doses of insulin has been reported to prevent diabetes in NOD mice and in BB rats adoptively transferred with spleen lymphocytes from acutely diabetic animals to their non-diabetic counterparts (Thivolet et al. (1991) Diabetologia 34, 314-319).
Such treatment may relieve the pancreatic beta-cells of metabolic demands and thus induce a state of "beta-cell rest". This quiescent state may be associated with diminished expression of many islet factors, including those that may serve as potential autoantigens at the cell surface (Aaen et al. (1990) Diabetes 39, 697-701; Kampe et al. (1989) Diabetes 38, 1326-1328). Non-specific immunostimulation caused by cytokine (Jacob et al. (1990) Proc. Natl. Acad. Sci. USA 87, 968-972) or adjuvant (Sadelain et al. (1990) Diabetes 39, 583-589) treatments, or environmental microbes have been implicated in other protocols of IDD prevention (Like et al. (1990) Diabetes 40, 259-262). A better understanding of the pathogenic role of insulin autoimmunity in IDD is clearly required.
There have been reports of efforts to induce antigen-specific immunoregulation to ameliorate autoimmune diseases (Steinman, L. (1990) Mol. Biol. Ned. 7, 333-339) . For example, various methods have been employed to induce antigen-specific suppression of experimental allergic encephalomyelitis (EAE) (PCT publication Wo 91/15225). Recently, several novel immunological approaches have been explored to autoimmune diseases such as EAE in mice and rats and lupus nephritis in MRL/l pr mice. Many have been directed toward blocking the function of the effector CD4.sup.+ T cell which has been shown to exhibit V.sub..beta. isotype restriction in EAE. These approaches have included the use of anti-TCR antibodies (Archa-Orbea et al., supra), synthetic TCR peptides (Offner et al. (1991) Science 251, 430-432) and superantigen treatment (Kim et al. (1991) J. Exp. Ned. 174, 1431-1437). The tolerogenic effects of enteral or pulmonal administration of antigens have also been described (Silverman et al. (1983) J. Immunol. 131, 2651-2661; Peng et al. (1990) Clin. Exp. Immunol. 81, 510-515; Michael (1989) Immune Invest. 18, 1049-1054; Kitamura et al. (1987) J. Immunol. 139, 3251-3259; Michalek et al. (1982) J. Immuno. 128, 1992-1998; Mowat et al. (1986) Immunol. 58, 677-683; PCT publications WO 91/12816, WO 91/01333; WO 92/06704). Nagler-Anderson et al. (1986) Proc. Natl. Acad. Sci. USA 83, 7443-7446 describe the suppression of arthritis by oral administration of soluble type II collagen in a collagen-induced arthritis mouse model. Deficiencies in this ability have been reported in several experimental mouse models of autoimmune diseases (Gesualdo et al. (1990) J. Inmaunol. 145, 3684-3691; Miller et al. (1984) Clin. Immunol. Immunopathol. 31, 231-240).
Zhang et al. have in NOD mice observed a reduced frequency of diabetes in response to very high doses of oral insulin (Zhang et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10252-10256) and further demonstrated in co-transfer studies that splenocytes from insulin-fed NOD mice prevented the adoptive transfer of diabetes by splenocytes from untreated diabetic mice to irradiated recipients.
In contrast, oral administration of insulin to spontaneously or induced diabetes-prone BB rats was reported not to offer any protection against diabetes (Mordes et al., 1995, The New York Academy of Science, "Oral Tolerance: Mechanism and Application").
Muir et al. (PCT publication WO 94/23737) have reported that, in NOD mice, administration of human insulin or its B-chain together with a powerful immune-stimulating adjuvant provides a small but significant protection against diabetes. This protection can be adoptively transferred to irradiated NOD mice co-infused with diabetogenic spleen cells and is associated with a reduced expression of gamma-interferon in the islet infiltrating lesion.
Also in the NOD mouse, nasal administration of insulin or a fragment of its B-chain has been reported to protect against diabetes adoptively transferred to non-diabetic NOD mice with a diabetogenic NOD T cell clone reactive against an amino acid sequence harboured in the insulin B-chain fragment referred to above or against spontaneous diabetes in NOD mice (Wegman et al. (1995) The New York Academy of Science, "Oral Tolerance: Mechanism and Application").
In yet another example of the use of insulin in the protection against diabetes, it has been reported that a single intravenous injection of insulin into NOD mice is followed by a reduced incidence of diabetes compared to non-treated animals.
The above examples of using insulin for preventing the onset of clinical diabetes have all been carried out in animal models of IDD, i.e. the NOD mouse and BB rat, but there is an increasing evidence that the same phenomena occur in the case of human IDD. Thus, fewer individuals, at high risk of developing IDD based on family history and immune markers, that were treated in the non-diabetic state with daily s.c. injections of insulin developed IDD than untreated individuals at a comparable high risk. Currently, large clinical studies in the US and in Europe are undertaken to formally prove whether prophylactic insulin therapy in non-diabetic, at-risk individuals can protect against subsequent development of IDD.
The mechanism whereby insulin exerts its effect in protecting against diabetes are not known exactly, but two issues are common to the above mentioned examples of the prophylactic use of insulin. One is that in most cases hormonally active insulin has been used in doses sufficient to cause constant or transient hypoglycaemia. In fact, the doses used in the animals studies have often led to high frequencies of deaths due to hypoglycaemia and the doses used in humans are of a magnitude where hypoglycaemic symptoms may frequently be expected.
The other common issue is the fact that a non-hormonal, immunologic protective mechanism of insulin, e.g., nasal administration of insulin B-chain fragment, relies on the use of a genetically homogenous animal strain. The immune response to an antigenic stimulus, whether of immunizing or tolerogenic nature, occurs via the presentation of a peptide fragment of the protein antigen in question to T cells. The peptide is presented on a cellular receptor, the MHC (major histocompatibility) molecules. The MHC molecules are extremely polymorphic, thus two classes of molecules exist, class I and class II and within each class several loci exist, e.g., the HLA class I (A, B and C loci) and HLA class II (DR, DQ and DP) in humans, and within each locus a large number of polymorphic alleles exist. Different MHC molecules present different peptides to T cells, even from the same antigen. Consequently, to obtain a T cell response, be it immunogenic or tolerogenic, to a given antigen, e.g., insulin in an outbred, MHC polymorphic population like humans, administration of the entire antigen, or at least the major part of it is likely to be required to obtain the desired response in all or most individuals treated.
According to the present invention, an insulin-like polypeptide without hypoglycaemic effect is used to prevent against the development of diabetes. The insulin is rendered hormonally inactive by discrete, specified deletions or amino acid substitutions which leaves the insulin minimally changed or as a fragment of the endogenous molecule. The present invention makes possible the treatment of clinical healthy individuals, albeit at a certain risk of developing a serious disease, with a disease specific antigen, insulin, without exposing the individual to the potential serious adverse effects, hypoglycaemia and mitogenicity secondary to the actions of hormonally active insulin. In an example of the present invention, daily sc injections with human insulin with a single amino acid substitution in position B25 that renders the molecule less than 0.1% hormonally active, in NOD mice protects as well against diabetes as equimolar doses of human insulin does, but without the adverse effects of insulin, i.e., approx. 50% deaths due to hypoglycaemia.